Liquid chromatography (LC) columns have been extensively developed and are used routinely in both analytical and preparative chromatography. The separation in a chromatography column of a sample comprising a mixture of components (also termed analytes or solutes) is achieved by dissolving the sample if it is solid in a liquid and injecting the sample into a flowing stream of mobile phase. The sample may be a liquid in which case dilution with another liquid prior to injection is optional. The components of the sample are carried through a tubular column where they may interact with the stationary phase typically packed within, thereby causing the sample to separate into its components due to different partitioning between the mobile and stationary phases of the different components (i.e. the components have different partition coefficients). In liquid chromatography the stationary phase is typically in the form of a bed of particles packed within the column.
The primary advantage of LC stems from its ability to separate, determine the concentration of and identify (with suitable means) 60-80% of all existing compounds. Column liquid chromatography is the most commonly used type of LC. The separation of components of a mixture as the mixture passes through the column is based on each type of chemical constituent having a unique velocity. The velocity of a constituent depends on its partitioning between the moving liquid passing through the column and the particles that make up the so-called packed bed in the column. In column chromatography when an analytical measurement is the objective, e.g. measure component concentrations or determine what components are present, a sample with a volume much smaller than the column volume is introduced to the column. The sample when first introduced into the column thus occupies a small zone containing all of its components at the head or top of the column. As the separation proceeds, the components separate based on differences in partitioning, but the zones also spread due to a number of processes occurring within the column that are well-known to one of skill in the art. A separation is better when these natural spreading processes are minimized. The recent significant improvements in particle technology (i.e. sub-2 μm fully porous and core-shell particles) provide scientists with better separations by minimizing the natural spreading process.
Small volume samples are commonly encountered in the fields of metabolomics, proteomics, forensics, neurochemistry and single cell analysis. The high complexity and mass limited nature of such small samples requires the column to have a small diameter to limit sample dilution. On the other hand, injecting a volume that is too small leads to difficulty in making quantitative measurements because the components are dilute at the time they are detected leading to a low analytical signal. Injecting a volume that is too large makes the initial zone on the top of the column wider, adding to the natural spreading occurring on column, making the separation worse. The latter problem is called volume overload. In many critical applications, the range of allowable injection volumes is small. The injection volume must be large enough to provide an adequate signal and be small enough to avoid significantly increasing the spreading of the zones.